WRKY27-<i>SPDS1</i> module of Ichang papeda (<i>Citrus ichangensis</i>) promotes cold tolerance by modulating spermidine content

Spermidine (Spd) is one of the predominant polyamines in higher plants and plays a crucial role in combating various abiotic stresses. However, the molecular functions and underlying regulatory mechanisms associated with plant Spd synthase (SPDS) genes in cold tolerance remain poorly understood. In this study, cold treatment markedly induced Spd accumulation and enhanced SPDS activity in Ichang papeda (<i>Citrus ichangensis</i>), a cold-hardy plant in <i>Citrus</i> genus. Exogenous Spd supply led to dramatically improved cold tolerance. Two SPDS genes (<i>CiSPDS1</i> and <i>CiSPDS2</i>) were identified in Ichang papeda, but only <i>CiSPDS1</i> was substantially upregulated by cold. Overexpressing of <i>CiSPDS1</i> in both tobacco (<i>Nicotiana tabacum</i>) and lemon (<i>Citrus limon</i>), a cold-sensitive <i>Citrus</i> species, promoted Spd synthesis and enhanced cold tolerance in the transgenic plants. In contrast, knockdown of <i>CiSPDS1</i> in Ichang papeda by virus-induced gene silencing (VIGS) repressed Spd synthesis and greatly impaired the cold tolerance, which was restored by exogenous replenishment of Spd. In addition, we demonstrated that WRKY27 of Ichang papeda (CiWRKY27) directly bound to and activated the <i>CiSPDS1</i> promoter through interacting with a W-box <i>cis</i>-acting element. Meanwhile, VIGS-mediated silencing of <i>CiWRKY27</i> resulted in marked reduction of <i>CiSPDS1</i> transcript levels and Spd contents and significantly impaired the cold tolerance in Ichang papeda. Taken together, this study illustrated the role of <i>CiSPDS1</i> in cold tolerance and identified it as a direct target of CiWRKY27. These findings provide insight into the regulatory mechanism by which the molecular module CiWRKY27-<i>CiSPDS1</i> regulates Spd accumulation for modulation of cold tolerance.